Recent progress in biosciences resulted in redirecting of research interests to a large extent from individual bio-molecules to the problems how these biomolecules are organized in more complex structures and how these structures function in the living cell. Extensive experience of working with individual biomolecules resulted in the development of numerous highly efficient techniques for the isolation and purification of molecular objects with molecular weights less than 106 Da. Contrary, the purification of larger objects, often combined under the name of nanoparticles, like plasmids, cell organelles, viruses, protein inclusion bodies, macromolecular assemblies as well as the separation of cells of different kind still remains a challenge. Large particle sizes (100-1000 nm), low diffusion rates, and complex molecular surfaces distinguish such objects from protein macromolecules (commonly <10 nm).
Traditionally used approaches for isolation of nanoparticles, as ultracentrifugation and micro/ultrafiltration are limited either in scale or resolution due to the similarities of size and density of cell debris and target nanoparticles. Partitioning in aqueous two-phase systems could be used alternatively for the isolation of nanoparticles but it suffers from the necessity to separate the target product from the phase-forming polymer.
Selective adsorption to a chromatographic matrix is a method, which offers many potential advantages with respect to resolution scale-up and process integration. It is noteworthy that only a small number of commercial chromatographic matrixes such as Sephacryl S-1000 SF from Amersham Pharmacia are claimed to accommodate spherical particles up to 400 nm in diameter within the intra-particle pores.
Nanoparticles and cells have very low diffusion coefficients due to the large size and they could be forced inside the pores only by a convective flow. For beaded chromatographic matrices most of the convective flow in the column goes through the voids in between the beads. Even for recently developed superporous beads with pore size of 800 nm up to 95% of the flow goes through the voids around the beads.
In early 90-s Svec, F. and Fréchet, J. M., Science 273:205-211 (1996), suggested to use molded continuous chromatographic media or so called macroporous monoliths, produced by the controlled polymerization inside the chromatographic column. Typically these monoliths are produced by polymerization of styrene or acrylate monomers and contain flow-through pores with diameters in the range of 700-2000 nm (0.7-2 μm). Later on, continuous superporous chromatographic media with pores as large as 20-200 μm were produced from agarose by Gustavsson, P. E. and Larsson, P-O., J. Chromatog. A. 795:199-210 (1998); Braas, G M F, et al., Trans. Inst. Chem. Eng. 78:11-15 (2000). These pores could easily accommodate objects as large as yeast cells.
Cryogels have appeared recently as a new class of materials with a combination of unique properties. Highly porous polymeric materials with a broad variety of morphologies could be produced from practically any gel-forming precursors using cryotropic gelation technique. Cryotropic gelation (cryogelation or cryostructuration are often used synonyms) is a specific type of gel-formation which takes place as a result of cryogenic treatment of the systems potentially capable of gelation. [Lozinsky, V. I., Vainerman, E. S., Rogozhin, S. V., Method for the preparation of macroporous polymer materials, SU Inventor's Certificate No. 1008214 (1982).] The essential feature of cryogelation is compulsory crystallization of the solvent, which distinguishes cryogelation from chilling-induced gelation when the gelation takes place on decreasing temperature e.g. as gelation of gelatine or agarose solutions which proceeds without any phase transition of the solvent.
The processes of cryogelation have some unique characteristics.
1. Cryotropic gel formation is a process which proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. At moderate temperatures below the freezing point some of the liquid remains still non-frozen accumulating in high concentrations (so called cryoconcentrating) all the solutes present in the initial solution. Chemical reactions or processes of physical gelation proceed in the non-frozen microphase at apparently much higher concentrations than in the initial.
2. The result of cryoconcentrating of dissolved substances in non-frozen liquid is a decrease in the critical concentration of gelation as compared to traditional gelation at temperatures above the freezing point.
3. Usually cryogelation in moderately frozen samples proceeds faster than traditional gelation at temperatures above the freezing point.
4. Frozen crystals of the solvent play a role of porogen when cryogels are formed producing a system of interconnected macropores. The macropore size could be as large as a few hundreds μm (Ø). The cryogels have often sponge-like morphology contrary to continuous monophase traditional gels produced from the same precursors at temperatures above freezing. Most of the solvent in cryogels is capillary bound and could be easily removed mechanically.
5. Temperature dependence of cryogelation has usually an optimum due to the balance between the effects facilitating gelation (cryoconcentrating) and factors decelerating it (low temperature, high viscosity in liquid microphase).
6. Cryogels are mechanically strong, but non brittle due to the elasticity of polymer walls in between macropores.
7. The porosity, mechanical strength and density of cryogels could be regulated by the temperature of cryogelation, the time a sample is kept in a frozen state and freezing/thawing rates.
The production of cryogels in general is well documented. For a review, vide e.g. Kaetsu, I., Adv. Polym. Sci. 105:81 (1993); Lozinsky, V. I. and Plieva, F. M., Enzyme Microb. Technol. 23:227-242 (1998); and Hassan, Ch. M. and Peppas, N. A., Adv. Polym. Sci. 151:37 (2000).
The most intensely studied cryogels are those prepared from poly(vinyl alcohol) (PVA) due to their easy availability. Thus when cooling an aqueous solution of PVA to a temperature within a range below 0° C. the ratio between gelling of the PVA and the crystallization of water is such that cryogels are easily formed. However, the preparation of cryogels by polymerizing an aqueous solution of monomers under chilling to a temperature below 0° C., at which water in the system is partially frozen with the dissolved substances concentrated in the non-frozen fraction of water, to the formation of a cryogel is also disclosed in literature. Thus, the preparation of cryogels by polymerizing an aqueous solution of acrylamide and N,N′-methylene-bis-acrylamide in the presence of a radical polymerization initiator under chilling to a temperature below 0° C. is disclosed, e.g. by E. M. Belavtseva et al., Colloid & Polymer Sci. 262:775-779 (1984); V. I. Lozinsky et al., Colloid & Polymer Sci. 262:769-774 (1984) and D. G. Gusev et al., Eur. Polym. J. Vol. 29, No. 1, 49-55, 1993. However, these references only report on studies of the cryostructurization of this polymer system and no practical use for the cryogels is suggested therein.
Further cryogels prepared by polymerizing an aqueous solution of monomers under chilling at a temperature at which solvent in the system is partially frozen with the dissolved substances concentrated in the non-frozen fraction of the solvent is disclosed in SU Inventor's Certificate No. 1008214. However, no use of the cryogels as a separation medium is disclosed therein.
It is an object of the present invention to provide new macroporous gels which can be used as a separation medium for chromatographic separations and separations in an electrical field.
It is another object of the present invention to provide macroporous gels prepared by polymerizing one or more monomers in solution under freezing to a temperature below the solvent crystallization point and having improved properties due to the presence of specific additives in said solution.
It is still another object of the present invention to provide macroporous gels prepared by polymerizing one or more monomers in solution under freezing to a temperature below the solvent crystallization point and having been modified to exhibit properties particularly suited for their use as a separation medium.
It is another object of the present invention to provide macroporous gels which can be used for the separation of cells, viruses or nanoparticles from suspensions thereof.
It is a further object of the present invention to provide macroporous gels which can be used for the separation of cells from a cell mixture according to specific properties of their surface.
It is also an object of the present invention to provide macroporous gels which can be used for adsorption affinity chromatography of microbial cells carrying a metal-binding peptide expressed at their surface.
It is still a further object of the present invention to provide macroporous gels which can be used for the separation of low-molecular weight products or proteins from a cellular suspension or crude homogenate according to the charge, hydrophobicity or affinity of the products or proteins.
These and other objects are attained by means of the present invention.